Disentangling the molecular determinants for Cenp‐F localization to nuclear pores and kinetochores. (9th April 2018)

• Record Type: Journal Article
• Authors: Berto, Alessandro; Yu, Jinchao; Morchoisne‐Bolhy, Stéphanie; Bertipaglia, Chiara; Vallee, Richard; Dumont, Julien; Ochsenbein, Francoise; Guerois, Raphael; Doye, Valérie
• Is Part Of: EMBO reports. Volume 19:Number 5(2018)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2018-04-09
• Subjects: Cenp‐F -- in silico modeling -- kinetochores -- mitosin -- nuclear pore; Molecular biology -- Periodicals; Molecular Biology -- Periodicals; Molecular biology; Periodicals; 572.8
• DOI: 10.15252/embr.201744742
• Languages: English
• ISSNs: 1469-221X

Abstract

Abstract: Cenp‐F is a multifaceted protein implicated in cancer and developmental pathologies. The Cenp‐F C‐terminal region contains overlapping binding sites for numerous proteins that contribute to its functions throughout the cell cycle. Here, we focus on the nuclear pore protein Nup133 that interacts with Cenp‐F both at nuclear pores in prophase and at kinetochores in mitosis, and on the kinase Bub1, known to contribute to Cenp‐F targeting to kinetochores. By combining in silico structural modeling and yeast two‐hybrid assays, we generate an interaction model between a conserved helix within the Nup133 β‐propeller and a short leucine zipper‐containing dimeric segment of Cenp‐F. We thereby create mutants affecting the Nup133/Cenp‐F interface and show that they prevent Cenp‐F localization to the nuclear envelope, but not to kinetochores. Conversely, a point mutation within an adjacent leucine zipper affecting the kinetochore targeting of Cenp‐F KT‐core domain impairs its interaction with Bub1, but not with Nup133, identifying Bub1 as the direct KT‐core binding partner of Cenp‐F. Finally, we show that Cenp‐E redundantly contributes together with Bub1 to the recruitment of Cenp‐F to kinetochores. Synopsis: Cenp‐F contributes to multiple processes throughout the cell cycle. Here, key residues required for Cenp‐F recruitment to nuclear pores or kinetochores are identified using in silico structural modelling, yeast two‐hybrid assays and localization studies. A conserved helix within the Nup133 β‐propeller interacts with a leucine zipper‐containing dimeric segment of Cenp‐F. The direct interaction with Bub1 is important for Cenp‐F recruitment to kinetochores. Mutations within the Cenp‐F C‐terminus discriminate NPC/nuclear bodies from kinetochore targeting. Abstract : Cenp‐F contributes to multiple processes throughout the cell cycle. Here, key residues required for Cenp‐F recruitment to nuclear pores or kinetochores are identified using in silico structural modelling, yeast two‐hybrid assays and localization studies.